title: Functional impact of colorectal cancer-associated mutations in the transcription factor E2F4.
abstract: The transcription factor E2F4 plays a critical role in cell cycle progression of normal and cancerous intestinal epithelial cells. Contrary to other E2Fs, the coding region of the E2F4 gene contains a longer spacer segment of a CAG trinucleotide repeat sequence encoding 13 consecutive serine residues, which is highly vulnerable to frameshift mutations in situations of genetic instability. Mutations in this region of the E2F4 gene have been observed in colorectal tumors with microsatellite instability. However, the effect of these changes on its function in colorectal cancer cells is currently unknown. We generated E2F4(CAG)₁₂ and E2F4(CAG)₁₄ mutants and compared their activity to the E2F4 wild-type, E2F4(CAG)₁₃. Luciferase assays with the thymidine kinase-luc reporter gene revealed that the mutants were more transcriptionally active than wild-type E2F4. The mechanism of increased activity of E2F4 was primarily related to protein stability, due to a significantly enhanced half-life of E2F4 mutants comparatively to that of wild-type E2F4. However, the association with the pocket protein p130/RBL2 did not account for this increased protein stability. Sequencing analysis of the endogenous E2F4 gene in a series of colorectal cancer cell lines showed that the microsatellite-unstable cell line SW48 exhibited a serine deletion in this gene. Accordingly, E2F4 half-life was much more elevated in SW48 cells in comparison to Caco-2/15, a microsatellite-stable cell line. Notably, in soft-agar assays, both mutants more potently increased anchorage-independent growth in comparison to wild-type E2F4. In conclusion, our data demonstrate that cancer-associated E2F4 mutations enhance the capacity of colorectal cancer cells to grow without anchorage, thereby contributing to tumor progression.
authors.affiliation: Department of Anatomy and Cellular Biology, Cancer Research Pavillon, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec J1E4K8, Canada.
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Paquin
authors.forename: Caroline
authors.initials: C
authors.lastname: Leblanc
authors.forename: Etienne
authors.initials: E
authors.lastname: Lemieux
authors.forename: Benjamin
authors.initials: B
authors.lastname: Bian
authors.forename: Nathalie
authors.initials: N
authors.lastname: Rivard
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D018938:Caco-2 Cells; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015179:Colorectal Neoplasms / Q000235:genetics*; D050704:E2F4 Transcription Factor / Q000235:genetics*; D045325:HCT116 Cells; D057809:HEK293 Cells; D019073:HT29 Cells; D006801:Humans; D053842:Microsatellite Instability; D009154:Mutation / Q000235:genetics; D050717:Retinoblastoma-Like Protein p130 / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic / Q000235:genetics; D018911:Trinucleotide Repeats / Q000235:genetics
chemical_list: D050704:E2F4 Transcription Factor; C493762:E2F4 protein, human; C493837:RBL2 protein, human; D050717:Retinoblastoma-Like Protein p130